Primary cells derived from first-trimester rhesus monkey (Macaca mulatta) placenta were transformed with a DNA fragment consisting of the early region of the SV40 genome to become a T-antigen-positive cell strain with numerous characteristics of trophoblast epithelium. The cell strain, termed RheTro, appears to be a useful in vitro analog to normal rhesus trophoblast cells. An examination of various rhesus monkey (Macaca mulatta) organs has shown type C viral antigen expression preferentially in the placenta (Stromberg, K. and Huot, R., Virology 122: 365-369, 1981). Separate cocultivations of isolated primary trophoblasts from ten rhesus monkey placentas with cell lines from heterologous mammalian species led to rapid isolation of type C rhesus retroviruses in four of ten cases. These four retrovirus isolates have been designated MMC-2 through MMC-5. Distinction of these viral isolates from the initial rhesus isolate (MMC-1) and the previous isolate from the stumptail monkey, Macaca arctoides, (MAC-1) could be made by host range studies and liquid DNA hybridization, but not by limited restriction endonuclease digestion. Specifically, the cellular DNA from rhesus isolates MMC-2 through MMC-5 melted 0.7 degree C to 1.0 degree C lower than either MAC-1 or MMC-1. Using our hybridization conditions, it was not possible to distinguish between MAC-1 and the originally reported isolate from rhesus,MMC-1. Both MAC-1 and MMC-1 were obtained in single, long-term cocultivation experiments (over seven months). The present isolates, MMC-2 through MMC-5, were detected in two to five weeks. The prompt detection of type C particle expression following cocultivation of rhesus trophoblast cells with heterologous cell lines demonstrates that trophoblast, as a differentiated cell type, is relaxed for complete retroviral expression. It remains to be seen if other primate species, including higher apes and man, will ylield infectious retrovirus using this approach.